06-methylguanine-DNA methyltransferase (MGMT), which is responsible for repair of the promutagenic and probably procarcinogenic 06-methylguanine produced in DNA by simple methylating carcinogens and mutagens, is tightly regulated both in bacteria and mammals. In mammalian tissues and cell lines the level of MGMT varies widely and the enzyme activity is lost in many cell lines transformed with virus or chemicals, and in some cases in apparently untransformed cell lines. In order to elucidate the mechanism of regulation of the enzyme, attempts will be made to clone the cDNA of the methyltransferase from human cells and to isolate the genomic clone of the enzyme. The procedure involves purification of the enzyme to near homogeneity from human placenta and the purified enzyme will be used to prepare monoclonal antibody. A cDNA library of the MGMT+ HeLa cells (containing 2x100,000 enzyme molecules/cell) will be prepared in bacterial cloning vectors, subsequent to a possible enrichment of poly(A)+RNA with the monoclonal antibody. The MGMT cDNA clone will be identified in E. coli colonies transformed with the cDNA library by differential hybridization with (P32) cDNA probes from poly(A)+RNAs of MGMT+ and MGMT- HeLa cells. The identity of the cDNA clone will be confirmed by using it to purify MGMT mRNA, and the latter's subsequent in vitro translation to produce MGMT identified by its antibody precipitation, size and pI. The cloned MGMT cDNA will be physically characterized and will be used (1) to isolate and to characterize the human genomic clone, (2) to study regulation of transcription of the MGMT gene in MGMT+ and MGMT- human cells, (3) as a probe to isolate the rat MGMT cDNA clone for use in studying regulation of expression of MGMT in different rat tissues and cell lines, (4) to map the MGMT gene in human and mouse chromosomes, and (5) for cloning in E. coli and mammalian expression vectors and possible large-scale synthesis of human MGMT in E. coli as a native or fusion protein. The human and rat MGMT cDNAs will be sequenced for comparison of their predicted amino acid sequences. The flanking regions of MGMT genomic clones from rat tissues having different levels of MGMT activity will be examined for changes which might explain differences in transcription. In parallel with the above studies, a survey of MGMT in various interspecies hybrids of MGMT+ and MGMT- cells may lead to chromosomal localization of a structural and/or regulatory locus of MGMT.